Relative natriuretic activities of synthetic atriopeptins I, II and III

Recent evidence indicates that mammalian atria contain a series of peptides which possess potent natriuretic activity. Using a sensitive and reproducible bioassay developed by this laboratory, the natriuretic and diuretic activities of three peptides, atriopeptins I, II, and III (21, 23 and 24 amino acids, respectively) were compared. Bioassays were conducted in pentobarbital-anesthetized male Sprague-Dawley rats weighing 300-350 grams. At doses ranging from 0.33 to 3.0 micrograms, no significant differences in natriuretic or diuretic activities were observed between the three peptides. The time courses of the natriuretic and diuretic responses to these peptides were also identical. The finding that atriopeptin I (21 amino acids) possesses natriuretic activity equal to that of atriopeptins II and III suggests that the C-terminus residues of atriopeptin III (Phe-Arg-Tyr) are not necessary for full expression of natriuretic activity. However, since several previous reports have indicated that atriopeptin I is considerably less potent as a natriuretic than we report here, perhaps a cautionary note should be sounded concerning the conditions required to produce or to retain full biologic activities of the synthetic atriopeptins.     

Electrophysiological study of the effects of cardiodilatin 1-16 on the frog spinal cord in vitro

Using the isolated spinal cord of the frog, hemisected and further divided into two distinct quadrants, we studied electrophysiological changes produced by peptides present in the atrial natriuretic factor (ANF) preprohormone. ANF and related peptides (atriopeptin I and atriopeptin III) did not affect the frog spinal cord. The 1-16 fragment from cardiodilatin (10(-5) M) induced slow depolarization in ventral and dorsal nerve stumps. The depolarization was associated with an increase of the evoked dorsal root potentials and depression of the fast component of the reflex responses. When depolarization approached its maximum value, spontaneous slow potentials appeared progressively similar to the evoked potentials, and became rhythmic until they reached a frequency of one potential every 15-20 seconds. The effects of cardiodilatin 1-16 are localized at dorsal horn level. It is suggested that this substance exerts a modulatory effect on frog cord physiology.     

Atrial dipeptidyl carboxyhydrolase is a zinc-metallo proteinase which possesses tripeptidyl carboxyhydrolase activity

Atrial dipeptidyl carboxyhydrolase readily converts one atrial natriuretic peptide, atriopeptin II (Ser103-Arg125 peptide), to another, atriopeptin I (Ser103-Ser123 peptide), by selective removal of the C-terminal dipeptide, Phe-Arg. The atrial peptides possess natriuretic, diuretic, smooth muscle relaxant, and cardiodynamic properties and their existence has shown the mammalian heart to be an endocrine organ. After inactivating the bovine atrial enzyme with EDTA, activity is restored by the addition of Co+2, Zn+2 and Mn+2 but not by Cu+2, Mg+2, Ca+2, or Cd+2. The enzyme is thus likely to be a zinc-metallo proteinase. In addition to its dipeptidyl activity, the enzyme also displays tripeptidyl carboxyhydrolase activity with atriopeptin III (Ser103-Try126 peptide) as substrate. The hydrolytic products resulting from tripeptidyl cleavage are atriopeptin I and Phe-Arg-Tyr. However, with [mercaptopropionyl105,(D)Ala107]-atriopeptin III-NH2 peptide (a potent agonist of atriopeptin III) as substrate, the enzyme acts exclusively as a tripeptidyl carboxyhydrolase. To examine the basis for this shift in cleavage point, pentapeptides based on the C-terminal sequence of atriopeptin III were prepared; a C-terminal Tyr or Tyr-NH2 residue is not sufficient to cause the change in cleavage point. The amidated pentapeptide is not a substrate but is a competitive inhibitor of hydrolysis of the corresponding free-acid peptide.     

Effect of indomethacin and propranolol on the blood pressure and renin response to atriopeptin III in conscious rats

The effect of prostaglandin synthesis inhibition and of beta-adrenoceptor blockade on the blood pressure and renin response to the synthetic atrial natriuretic peptide atriopeptin III was assessed in unanesthetized normotensive rats. This peptide was infused i.v. for 30 min at a rate of 1 microgram/min in rats pretreated either with indomethacin (5 mg i.v.) or propranolol (1 mg i.v.). The blood pressure reducing effect of atriopeptin III was attenuated neither by indomethacin nor by propranolol. Atriopeptin III per se did not modify plasma renin activity. Both the administration of indomethacin and of propranolol had a suppressing effect on renin release during atriopeptin III infusion. These data suggest that the vasodilating properties of atrial natriuretic peptides do not depend in the conscious normotensive rats on the production of prostaglandins. They also provide evidence that during infusion of such peptides, both prostaglandins and beta-adrenergic mechanisms are still involved in the regulation of renin secretion.     

Prostaglandin E2 and atriopeptin III oppose the contractile effect of angiotensin II in rat kidney mesangial cell cultures

Effects of vasoconstrictory and of dilatory hormones were studied on the contractile activity of cultured rat kidney mesangial cells. By phase contrast microscopy, a rapid contraction was seen of most cells treated with angiotensin II (10(-6) - 10(-10) mol/L), which was sometimes followed by autonomous relaxation after 10 to 20 min. Prostaglandin E2 and atriopeptin III prevented the contractile effect of angiotensin II in a dose-dependent manner. Angiotensin II, but not atriopeptin III, stimulated prostaglandin E2 synthesis in mesangial cell cultures.     

Comparison of binding and cyclic GMP accumulation by atrial natriuretic peptides in endothelial cells

Rat 125I-labeled atrial natriuretic factor (ANF (8-33)) was used to identify ANF receptors on cultured bovine aortic endothelial cells. Specific binding of 125I-ANF at 37 degrees C to confluent endothelial cells was saturable and of high affinity. Scatchard analysis of the equilibrium binding data indicated that endothelial cells contain a single class of binding sites with a Kd of 0.1 +/- 0.01 nM. This particular clone of endothelial cells had 16000 +/- 1300 receptors per cell. The order of potency for competing with 125I-ANF binding was human atrial natriuretic peptide (hANP) = atrial natriuretic factor (ANF (8-33)) greater than atriopeptin II greater than atriopeptin III greater than atriopeptin. The weakest competitor, atriopeptin I, had a K1 of 0.45 nM, which was only 6-fold higher than the K1 for hANP and ANF (8-33). ANF (8-33) and hANP in the presence of 0.5 mM isobutylmethyl-xanthine produced a 15-20-fold increase in cyclic GMP content at 10 pM and a maximal 500-fold elevation of cyclic GMP at 10 nM. The concentrations required to elicit a half-maximal increase in cyclic GMP for hANP, ANF (8-33), atriopeptin I, atriopeptin II and atriopeptin III were 0.30, 0.35, greater than 500, 4.0 and 5.0 nM, respectively. Although atriopeptin I acted as a partial agonist, it was unable to antagonize the effect of ANF (8-33) on cyclic GMP formation. These findings suggest that endothelial cells have multiple and functionally distinct ANF-binding sites.     

Atriopeptins: a family of potent biologically active peptides derived from mammalian atria

Extracts of rat atria are potent stimulators of sodium and urine excretion, and relax vascular and intestinal smooth muscle preparations. The structures of six biologically active peptides obtained from atrial extracts are reported here. Ion exchange chromatography of a low molecular weight fraction obtained by gel filtration of atrial extracts produced two natriuretic fractions: the first induced relaxation of intestinal smooth muscle strips only, whereas the second also relaxed vascular strips as well. From the first fraction four pure biologically active peptides obtained by reverse phase HPLC have been sequenced: the 21 amino acid peptide, designated atriopeptin I, and three homologs (des- ser1 -, des- ser1 -ser2-, and des- ser21 - atriopeptin I). From the second fraction two pure biologically active peptides were obtained, which had C-terminal extensions of atriopeptin I: atriopeptins II (23 amino acid residues) and III (24 residues), having respectively phe-arg and phe-arg-tyr C-termini. These results suggest that this family of six peptides, sharing the same 17 membered ring formed by an internal cystine disulfide, is derived from a common high molecular weight precursor.     

Chronotropic and inotropic effects of atrial peptides on the isolated systemic heart ofOctopus vulgaris

Summary The chronotropic and inotropic effects of four atrial peptides (cardiodilatin 1–16, atrial natriuretic factor 8–33 and atriopeptin I and III) on the isolated systemic heart ofOctopus vulgaris were studied.Using a preparation that produces a physiological stroke volume at physiological input pressures, it was found that ANF, atriopeptin I and atriopeptin III exerted both negative chronotropic and inotropic effects. In contrast, cardiodilatin produced a positive inotropic effect.A dose-response curve of ANF is reported, showing a threshold concentration of about 10^–12 M.The pharmacological and physiological implications of these results are discussed in relation to some characteristics of the cephalopod systemic heart.     

Effect of cGMP-dependent signaling system in regulation of osmotic permeability in the frog urinary bladder

In frogs' isolated urinary bladders, contribution of cytosolic guanylate cyclase and cGMP-dependent protein kinase to regulation of osmotic permeability was studied. ODQ (25-100 microM), an inhibitor of cytosolic guanylate cyclase induced an increase of vasotocin-activated osmotic permeability but had no effect on the hormone-activated transepithelial urea transport. In isolated mucosal epithelial cells ODQ (50 microM) decreased the concentration of intracellular cGMP. In these cells L-NAME (0.5 nM), an inhibitor of NO synthase, also decreased the level of cGMP whereas cAMP was significantly increased. 8-pCPT-cGMP (25 and 50 microM), a permeable cGMP analogue which selectively activates protein kinase G, inhibited vasotocin-induced increase of water transport along osmotic gradient indicating that protein kinase G is involved in regulation of water reabsorption. The data obtained show that NO/cGMP signalling system in the frog urinary bladder appears to be a negative modulator of vasotocin-activated increase of osmotic permeability.     

The use of a monoclonal antibody to measure plasma atriopeptins in rat

A monoclonal antibody with specificity for atrial natriuretic peptides (ANP) was produced, that can be used for the radioimmunological determination of ANP-immunoreactivity (ANP-IR) in rat plasma. The antibody recognizes atriopeptin I, II, III, as well as alpha-hANP and alpha-hANP fragment (7-28) and does not crossreact with ANP-fragments (13-28) and (18-28). Plasma levels of ANP-IR in conscious Wistar rats were determined before and after volume-loading. Basal plasma levels of ANP-IR were 108 +/- 12 pg/ml, and after volume-loading increased to 800 +/- 59 pg/ml.     

Centrally administered atriopeptin III reduces water intake and vasopressin secretion in dehydrated sheep.

Two experiments were carried out to investigate the responses of sheep (N = 6) to intracerebroventricular (ICV) injections of atriopeptin III [atrial natriuretic factor (5-28), AP III]. In Experiment, 1, 24-h dehydrated animals were given 0 (saline vehicle control), 10 or 30 micrograms AP III directly before the presentation of water. The highest dose of AP III significantly (p less than 0.02) reduced the amount of water drunk in the subsequent 20 min. In Experiment 2, blood samples were taken at various intervals before and after ICV injection of 0 or 30 micrograms AP III when the sheep were water replete or 24-h dehydrated. Plasma concentrations of vasopressin (AVP) and cortisol were measured and estimates were made of plasma osmolality. In the dehydration condition, AVP levels were somewhat reduced (p less than 0.059) after AP III administration but no decrease was observed when the animals were euhydrated. No significant changes in plasma osmolality or cortisol concentrations were observed in response to AP III or the saline vehicle. Because a large dose of AP III (30 micrograms ICV) was required to produce the comparatively small behavioral and endocrine effects observed in this study, the results are suggestive of a pharmacological, rather than a physiological, action of the peptide in this species.     

In vivo atrial peptide-venodilation: minimal potency relative to nitroglycerin in dogs

Venodilation is thought to contribute to the hemodynamic actions of atrial peptides. Therefore, we measured the effective vascular compliance (EVC) as a parameter of overall venous tone in 7 pentobarbital anesthetized dogs under autonomic blockade during i.v. infusions of rat atriopeptin II (AP II, up to 100 pmol/kg/min), rat alpha-atrial natriuretic factor, and nitroglycerin (GTN). AP II lowered mean arterial pressure by reducing peripheral vascular resistance with a threshold between 3 and 10 pmol/kg/min (but was ineffective in anesthetized or conscious dogs without autonomic blockade). Neither atrial peptide altered EVC, while GTN augmented EVC and caused a 4.6-fold larger reduction of central venous pressure than AP II at equihypotensive dosage. These findings, with infusion rates probably close to endogeneous release, reveal a vasodilator potency of atrial peptides, which is restricted to systemic arterioles without affecting venous tone.     

Atriopeptin and spontaneous hypertension in rats

The involvement of atriopeptin in hypertension was investigated in spontaneously hypertensive rats (SHR). It was found that intravenous injection of atriopeptin III (20-80 nmol/kg) markedly decreased the mean arterial pressure in anesthetized SHR in a dose dependent manner. The heart rate was not significantly affected. The contents of atriopeptin immunoreactive material in the rat atrium and plasma were measured with radioimmunoassay. Both the atrium and plasma contents of atriopeptin immunoreactive material were found to be significantly higher in SHR than in the normotensive control Wistar Kyoto (WKY), indicating an increase in the biosynthesis and release of atriopeptin in SHR. Whether this change was a compensatory response induced by hypertension remains to be investigated.     

Dehydration attenuates the acute natriuretic response to rat atriopeptin III (rAPIII)

The acute natriuretic response to atrial peptides (AP) is highly variable in anesthetized rats, and some rats are unresponsive. To determine if this response to AP was affected by dehydration, we measured hematocrit, plasma volume, and natriuresis (delta UNaV) after intravenous injection of 3 micrograms/kg of rat atriopeptin III (rAPIII) in anesthetized rats deprived of water for 0, 12, 20, 29, 44, and 68 hours. Data were compared with those from rats receiving 1.5 mg/kg furosemide (FU) after 0 and 68 hours without water. There were 10- and 3-fold decreases in delta UNaV following rAPIII and FU injection after 20 and 68 hours without water, respectively. Hematocrit increased and plasma and total blood volumes decreased after 12 hours of dehydration. Plasma volumes and delta UNaV were correlated (r = 0.64, p less than 0.05; r = 0.75, p less than 0.001) in the combined groups receiving rAPIII (n = 30) and FU (n = 10), respectively. These results demonstrate that a relatively short period of water deprivation (WD) and the resulting hemoconcentration in rats decreased their acute natriuretic response to diuretics. Thus, differences in water intake may account for some of the large variation in delta UNaV after exogenous administration of rAPIII.     

Stimulatory effects of atrial natriuretic factor on phosphoinositide hydrolysis in cultured bovine aortic smooth muscle cells

The effects of atrial natriuretic factor (ANF) on phosphoinositide hydrolysis were examined in preparations of cultured bovine aortic smooth muscle cells. In homogenates or particulate fractions from cultured bovine aortic smooth muscle cells, ANF and atriopeptin I increased the formation of inositol phosphates and GTPase activity. The effects on inositol phosphates were markedly enhanced with guanosine 5'[gamma-thio]triphosphate. Both atrial peptides also stimulated the formation of diacylglycerol in intact cultured cells. In these experiments, atriopeptin I was about 10-fold more potent than ANF. These studies indicate that atrial peptides have stimulatory effects on phosphoinositide hydrolysis which are mediated through a guanine nucleotide regulatory protein. The greater potency of atriopeptin I on GTPase activity and the accumulation of inositol phosphates suggests that the nonguanylate cyclase-coupled receptor for ANF (ANF-R2) mediates the stimulatory effects of ANF on phosphoinositide hydrolysis through a guanine nucleotide regulatory protein.     

Steroidogenic effect of atrial natriuretic factor in isolated mouse Leydig cells is mediated by cyclic GMP.

The effects of different atrial natriuretic peptides on cyclic GMP formation and steroidogenesis have been studied in Percoll-purified mouse Leydig cells. Rat atrial peptides rANP (rat atrial natriuretic peptide), rAP-I (rat atriopeptin I) and rAP-II (rat atriopeptin II), in the presence of a phosphodiesterase inhibitor, stimulated cyclic GMP formation in a concentration-dependent manner. In the presence of saturating concentrations of the peptides, a 400-600 fold stimulation of cyclic GMP accumulation was observed. Among the peptides, rAP-II appeared to be the most potent. ED50 values (concentration causing half-maximal effect) for rAP-II, rANP and rAP-I were 1 X 10(-9) M, 2 X 10(-9) M and 2 X 10(-8) M respectively. A parallel stimulation of cyclic GMP formation and testosterone production by the cells was observed after incubation of the cells with various concentrations of rAP-II. In the presence of a saturating concentration of rAP-II (2 X 10(-8) M), maximum stimulation of intracellular cyclic GMP content was obtained within 5 min of incubation. Testosterone production by mouse Leydig cells could be stimulated by 8-bromo cyclic GMP in a concentration-related manner. At a 10 mM concentration of the cyclic nucleotide, steroidogenesis was stimulated to a similar extent as that obtained with a saturating concentration of human chorionic gonadotrophin (5 ng/ml). On the basis of these results we conclude that cyclic GMP acts as a second messenger in atrial-peptide-stimulated steroidogenesis in mouse Leydig cells. The steroidogenic effect of atrial peptides appears to be species-specific, since none of these peptides stimulated testosterone production by purified Leydig cells of rats, though in these cells a 40-60-fold stimulation of cyclic GMP formation in response to each of the three peptides was observed. However, 8-bromo cyclic GMP could stimulate testosterone production in rat Leydig cells. Therefore we conclude that the lack of steroidogenic response in rat Leydig cells to atrial-natriuretic-factor-stimulation results from an insufficient formation of cyclic GMP in these cells. This species difference would appear to result from a lower guanylate cyclase activity in rat Leydig cells.     

Atrial natriuretic factor and cGMP inhibit amiloride-sensitive Na+ transport in the cultured renal epithelial cell line, LLC-PK1

The renal cell culture model, LLC-PK1, which contains an amiloride-sensitive conductive Na+ transport pathway and a Na+/H+ exchanger, was utilized to examine the direct effects of atriopeptin II and cGMP on Na+ transport in epithelial cells. Exposure of cells to atriopeptin II (10(-7) M) increased cGMP production within 2 min of addition to cells in monolayer. Atriopeptin II (10(-7) M) or exogenous 8-bromo-cGMP (10(-3) M) maximally inhibited the uptake of 22Na+ through the conductive pathway which accounted for up to 60% of total 22Na+ uptake. The apparent Ki for this inhibition by atriopeptin II was 2 X 10(-11) M. Amiloride inhibited 22Na+ uptake to a similar extent as atriopeptin II, and the effects of the presence of both agents was not additive. In contrast, neither atriopeptin II nor cGMP blunted the increment in 22Na+ uptake induced by a pH gradient. Thus atriopeptin II can directly inhibit Na+ transport in renal epithelial cells, probably through its stimulation of cGMP.     

Effect of hemorrhage on plasma atriopeptin levels in conscious dogs

An increase in atrial pressure has been shown to cause an increase in the concentration of atrial peptides (atriopeptin) in plasma. We therefore hypothesized that a reduction in atrial pressure would decrease the concentration of atriopeptin in plasma. In formulating this hypothesis we assumed that changes in the concentration of other circulating hormones or changes in cardiac nerve activity during hemorrhage would not affect the secretion of atriopeptin. To test the hypothesis, we bled sham-operated conscious dogs at a rate of 0.8 ml.kg-1.min-1 to decrease right and left atrial pressures. Hemorrhage was continued until a total of 30 ml of blood per kilogram body weight had been removed. Identical experiments were performed on conscious cardiac-denervated dogs. The concentration of plasma atriopeptin was decreased in each group of dogs after 10 ml of blood per kilogram of body weight had been removed, but the decrease achieved statistical significance only in the cardiac-denervated dogs. Further hemorrhage, however, produced no further decreases in circulating atriopeptin in either group even though atrial pressures continued to decline as more blood was removed. A comparison of the atriopeptin response to hemorrhage revealed no significant difference between the sham-operated and cardiac-denervated dogs, thus providing no evidence for a specific effect of cardiac nerves on atriopeptin secretion during hemorrhage. Our results demonstrate that the relationship between atrial pressure and plasma atriopeptin that has been observed repeatedly during atrial stretch is not evident during relatively slow, prolonged hemorrhage. There is, however, a small decline in circulating atriopeptin during the initial stage of hemorrhage that could be of biological significance.     

Atriopeptin release from the isolated perfused rabbit heart

Mammalian atrial extracts have been shown to contain bioactive peptides which exert natruiretic, diuretic, and smooth muscle relaxant effects. These extracts include several low molecular weight (< 5,000 Mr) atrial peptides (atriopeptins) which exhibit identical sequences over a central core region which are derived from the high molecular weight peptide (atriopeptigen) precursor which has been purified and sequenced. In the current study we found that extracts of rabbit atria possess both high and low molecular weight bioactive atrial peptides, however, the coronary venous effluent obtained from the isolated perfused rabbit heart only contained the low molecular weight peptide. This trypsin labile activity causes a dose-dependent relaxation of rabbit aorta and chicken rectum assay strips. Separation of the bioactivity with gel filtration chromatography and reversed phase HPLC indicates the heart releases a single substance similar to atriopeptin III. There was no evidence that atriopeptigen was released from the isolated perfused rabbit heart. We suggest that atriopeptigen is proteolytically processed in the atria to an atriopeptin which is subsequently the released form of the atrial peptide.     

Phorbol and calcium decreased atriopeptin response in a human renal cell line.

We investigated regulation of atrial natriuretic factor (ANF)-stimulated cellular cGMP accumulation (ANF-s-cGMP) in an ANF-responsive human renal cell line, SK-NEP-1. Dose-response data indicated that the EC50 for ANF(99-126) was 1.1 x 10(-9) M. Brain natriuretic peptide (10(-6) M) increased cGMP to a level indistinguishable from that of ANF (10(-6) M). [Met-(O)]ANF was only half as potent as ANF, and atriopeptin I (10(-6) M) did not increase cGMP over basal levels. Preincubation of SK-NEP-1 cells with ANF, but not atriopeptin I (API), for two hours or longer, caused a concentration-dependent down-regulation of ANF-s-cGMP. Phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, and A23187 and its 4-bromo derivative, calcium ionophores, inhibited ANF-s-cGMP in a dose-dependent manner. A23187 inhibition was calcium dependent and promoted net cGMP degradation. Thirty-six hour preincubation with PMA, a procedure used to down-regulate PKC, abolished acute PMA inhibition of ANF-s-cGMP without having an effect on ANF-s-cGMP or on 4-bromo-A23187 inhibition thereof. These data indicate that PKC activation specifically inhibited ANF-s-cGMP but that PKC was not required for ANF-s-cGMP in SK-NEP-1 cells. Thus structurally related ANF peptides, protein kinase C (PKC) activators, calcium ionophores are potential modulators of ANF-s-cGMP in cells from this human renal cell line.